Concentration Recognition-Based Auto-Dynamic Regulation System (CRUISE) Enabling Efficient Production of Higher Alcohols

Abstract

Microbial factories lacking the ability of dynamically regulating the pathway enzymes overexpression, according to in situ metabolite concentrations, are suboptimal, especially when the metabolic intermediates are competed by growth and chemical production. The production of higher alcohols (HAs), which hijacks the amino acids (AAs) from protein biosynthesis, minimizes the intracellular concentration of AAs and thus inhibits the host growth. To balance the resource allocation and maintain stable AA flux, this work utilizes AA-responsive transcriptional attenuator ivbL and HA-responsive transcriptional activator BmoR to establish a concentration recognition-based auto-dynamic regulation system (CRUISE). This system ultimately maintains the intracellular homeostasis of AA and maximizes the production of HA. It is demonstrated that ivbL-driven enzymes overexpression can dynamically regulate the AA-to-HA conversion while BmoR-driven enzymes overexpression can accelerate the AA biosynthesis during the HA production in a feedback activation mode. The AA flux in biosynthesis and conversion pathways is balanced via the intracellular AA concentration, which is vice versa stabilized by the competition between AA biosynthesis and conversion. The CRUISE, further aided by scaffold-based self-assembly, enables 40.4 g L^-1 of isobutanol production in a bioreactor. Taken together, CRUISE realizes robust HA production and sheds new light on the dynamic flux control during the process of chemical production.

Keywords: amino acids; continue production; dynamic regulation; higher alcohols; self‐assembly.

Figure 1

Design of the CRUISE and the corresponding isobutanol production. a) The production modes. Ni: non‐induced; GiP: growth induced production; PiP: production induced production; GiPiG: growth induced production, production induced re‐growth, re‐growth induced re‐production. b) The pathways for AA biosynthesis and HA production. AlsS, acetolactate synthase; IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy‐acid dehydratase; LeuDH, leucine dehydrogenase; KivD, ketoisovalerate decarboxylase; YqhD, alcohol dehydrogenase; KIV, 2‐ketoisovalerate; KIC: 2‐ketoisocaproate. The CRUISE coupled AA‐concentration‐regulated ivbL transcriptional attenuation system and HA‐concentration‐regulated BmoR transcriptional activation system. For AA starvation, microbes closed the AA‐to‐HA conversion and biosynthesized AA via the endogenous AA biosynthesis pathway. For AA sufficiency, microbes turned on the AA‐to‐HA conversion. The generated HA bound to BmoR to enhance AA biosynthesis in a feedback activation mode, thereby enabling the continuous AA‐to‐HA conversion. c) The corresponding isobutanol production.

Figure 2

AA‐concentration‐regulated ivbL transcriptional attenuation system. a) The sequence of the ivbL transcriptional attenuation system. The ivbL transcriptional attenuation system is responsible for regulating the expression of acetylhydroxyacid synthase IlvBN. The polypeptide ivbL (96 bp) is located downstream of the promoter P_ivbL . b) Validation of the transcriptional attenuation of ivbL system toward L‐Val or L‐Leu. In the presence of L‐Val or L‐Leu feeding could inhibit the expression of GFP. The logic gate of “NOT GATE” was formed with L‐Val or L‐Leu as input and GFP as output. c) IvbL‐dependent inducible cascade activation system. In the presence of L‐Val or L‐Leu feeding could induce the expression of GFP. d) Screening of L‐Val overproducers using the inducible cascade activation system, and testing of the screening efficiency via FACS technique. Values and error bars represent mean and SD (n = 3), respectively. ^∗ P < 0.1, ^∗∗ P < 0.01, as determined by two‐tailed t‐test.

Figure 3

Concentration‐dependent unidirectional regulation in the CRUISE. a) The detailed biosynthetic pathways of isobutanol and isopentanol. IlvE, branched‑chain‑amino‑acid transaminase. b) Construction of the two‐layer cascade system to enable the precise inducible regulation of HA production by AA availability. c) The logic gate of “OR GATE” and the truth table of the system in (b). The input was AA or HA, and the output was GFP. d) The response effect of the system in b and the fluorescence intensity under fluorescence microscopy. e) Construction of the two‐layer cascade system to enable the precise inducible regulation of AA biosynthesis by HA availability. f) The logic gate of “OR GATE” and the truth table of the system in (e). The input was HA or AA, and the output was GFP. g) The response effect of the system in (e) and the fluorescence intensity under fluorescence microscopy. Values and error bars represent mean and SD (n = 3), respectively.

Figure 4

CRUISE‐driven continuous AA‐to‐HA conversion. a) The diagram of production and growth module mutually drove using the regulatory elements. b) The isobutanol production plasmid and the L‐Val biosynthesis plasmid. c) For isobutanol production, E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐B‐AII and pS‐iL‐LKY was used as the experimental strain, while E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐AII and pS‐LKY was used as the control strain. d) For isopentanol production, E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐B‐AII‐L and pS‐iL‐LKY was used as the experimental strain, while E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐AII‐L and pS‐LKY was used as the control strain. e) Fermentation of E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐B‐AII and pS‐iL‐LKY in nutrient‐rich M9Y medium. Scale‐up production in a 3‐L bioreactor. Values and error bars represent mean and SD (n = 3), respectively.

Figure 5

Continuous HA production reflected in transcription and expression levels. a) Differential gene analysis between the experimental strain and the control strain. E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐B‐AII and pS‐iL‐LKY was used as the experimental strain, while E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐AII and pS‐LKY was used as the control strain. The strains were cultured in yeast‐extract‐free M9NY medium. Volcano plot showed the number of differential genes between the experimental strain and the control strain at 60 h. The red and blue dots represented up‐ and down‐regulation, respectively. The horizontal coordinate represented the change of gene expression multiple in different samples, and the vertical coordinate represented the statistical significance of the difference in gene expression. Statistical map displayed the pathway distribution of differential genes. Rich factor refers to the ratio of the number of differential genes annotated in the pathway to the total number of annotated genes in the pathway. b) Heat map represented the differences in transcription levels of enzymes in the CRUISE and the L‐Val biosynthesis pathway. c) The amounts of GFP and RFP and the titer of isobutanol. Plasmid pS‐iL‐LKY‐G was used to characterize the expression of the enzymes in isobutanol production pathway. Plasmid pS‐B‐AII‐L was used to characterize the expression of the enzymes in L‐Val biosynthesis pathway. E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐B‐AII‐R and pS‐iL‐LKY‐G was used as the experimental strain, while E. coli MG1655ΔlacIZYA harboring plasmids pS‐AII‐R and pS‐LKY‐G was used as the control strain. Values and error bars represent mean and SD (n = 3), respectively.

Figure 6

Self‐assembly‐aided CRUISE to enable the dominant AA‐to‐HA conversion. a) The dominant production of isobutanol. The isobutanol production plasmid and the L‐Val biosynthesis plasmid. b) The structures of AlsS, CipA‐AlsS, IlvC, CipA‐IlvC, IlvD, CipA‐IlvD, and CipA‐IlvC‐IlvD were simulated by AlphaFold2. Pink represented CipA. Purple represented IlvC. Orange represented IlvD. c) The dominant production of isobutanol. 0 cipA represented E. coli MG1655ΔlacIZYA strain harbored plasmids pS‐B‐AII and pS‐iL‐LKY. 2 cipA represented E. coli MG1655ΔlacIZYA strain harbored plasmids pS‐B‐CA‐CIC‐ID and pS‐iL‐LKY. CipA was individually fused to AlsS and IlvC. 3 cipA represented E. coli MG1655ΔlacIZYA strain harbored plasmids pS‐B‐CA‐CIC‐CID and pS‐iL‐LKY. CipA was individually fused to AlsS, IlvC, and IlvD. 2 cipA‐F represented E. coli MG1655ΔlacIZYA strain harbored plasmids pS‐B‐CA‐CICD and pS‐iL‐LKY. CipA was individually fused to AlsS and the fusion IlvC‐IlvD. d) The dominant production of isopentanol. The isopentanol production plasmid and the L‐Leu biosynthesis plasmid. e) The dominant production of isopentanol. 0 cipA represented E. coli MG1655ΔlacIZYA strain harbored plasmids pS‐B‐AII‐L and pS‐iL‐LKY. 3 cipA represented E. coli MG1655ΔlacIZYA strain harbored plasmids pS‐B‐CA‐CIC‐ID‐CLA‐LBCD and pS‐iL‐LKY. CipA was individually fused to AlsS, IlvC, and LeuA. Values and error bars represent mean and SD (n = 3), respectively.

Figure 7

Utilization of HA‐tolerant strain for efficient isobutanol production. a) Verification of the isobutanol tolerance of different knockout strains and confirmation of the knockout region that determined the tolerance. b) Analysis of the correlation between the genes in the knockout region using PPI network from STRING database. c) The shake‐flask production of isobutanol using M9Y medium and the scale‐up production of isobutanol in a 3‐L bioreactor. For shake‐flask fermentation, E. coli MG1655ΔlacIZYA strain harboring plasmids pS‐B‐AII and pS‐iL‐LKY, E. coli MG1655Δ98k strain harboring plasmids pS‐B‐AII and pS‐iL‐LKY, and E. coli MG1655Δ98k‐2‐4 strain harboring plasmids pS‐B‐AII and pS‐iL‐LKY were used. For scale‐up fermentation, E. coli MG1655Δ98k‐2‐4 strain harboring plasmids pS‐B‐CA‐CICD and pS‐iL‐LKY was used. CipA was individually fused to AlsS and the fusion IlvC‐IlvD. Values and error bars represent mean and SD (n = 3), respectively. ^∗ P < 0.1, ^∗∗ P < 0.01, as determined by two‐tailed t‐test.