Employing a recombinant strain of Advenella mimigardefordensis for biotechnical production of Homopolythioesters from 3,3'-dithiodipropionic acid

Abstract

Advenella mimigardefordensis strain DPN7(T) was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3'-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of the buk-ptb operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from Clostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaC(Am)). These measures yielded the potent PMP production strain A. mimigardefordensis strain SHX22. The deletion of mdo was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne buk-ptb operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of A. mimigardefordensis (phaC(Am)) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.

Fig 1

Predicted DTDP degradation and PMP production pathway. (A) Inherent DTDP degradation pathway of A. mimigardefordensis. (B) DTDP-to-PMP conversion pathway in the engineered strain. The double box indicates the A. mimigardefordensis cell. The deletion of mdo is indicated with a bold X. Dashed arrows across the periplast indicate transmembrane transport of relevant molecules. The bold arrow indicates the overexpression of phaC_Am in plasmid pBBR1MCS5:: phaC_Am. All reactions catalyzed by enzymes are numbered. Bold A, B, and C indicate three important manipulations for improved PMP production. The detailed genetic operations of these three steps are given at the bottom. The buk-ptb operon originates from C. acetobutylicum and is regulated under its native promoter. Other genes in this figure originate from A. mimigardefordensis DPN7 and are regulated under their own promoters. 3MP, 3-mercaptopropionic acid; DTDP, 3,3′-dithiodipropionic acid; 3SP, 3-sulfinopropionic acid; lpdA, disulfide reductase; mdo, thiol dioxygenase; sucCD, succinyl-CoA synthetase; caiA, acyl-CoA dehydrogenase; buk, butyrate kinase; ptb, phosphotransbutyrylase; phaC_Am, poly(hydroxyalkanoic acid) synthase; caiB, acyl-CoA transferase; ahpD, alkylhydroperoxidase; rdgC, recombination-associated protein; recA, recombination protein; recX, recombination regulator protein; cfa, cyclopropane-fatty-acyl-phospholipid synthase; phaB, acetoacetyl-CoA reductase; phaR, polyhydroxyalkanoate synthesis repressor protein; PphaC_Am, native promoter of phaC_Am.

Fig 2

Growth of different strains in MS medium containing sodium gluconate or DTDP. All four strains were incubated in different media. (A) MS medium with 5 g/liter sodium gluconate. (B) MS medium with 5 g/liter DTDP. The OD₆₀₀ was set to 0.02 after inoculation and before growth started. OD₆₀₀ profiles of different samples were measured in daily intervals.

Fig 3

PMP production of indicated strains in MS medium using sodium succinate as the carbon source. All indicated strains were first incubated in MS medium containing 5 g/liter sodium succinate as the carbon source. In the late exponential phase, all of them were transferred into fresh medium containing 5 g/liter DTDP. After 4 days of incubation, PMP contents from different samples were analyzed.

Fig 4

PMP production in A. mimigardefordensis SHX5 and SHX9 by using different carbon sources. The cells were first incubated in MS medium containing different carbon sources at a concentration of 5 g/liter except sodium propionate, which was supplied at a concentration of only 2 g/liter, in the first cultivation stage. When sodium gluconate, sodium propionate, or sodium succinate was used as the carbon source, the weight of sodium was eliminated. In the late exponential phase, all cells were transferred into the same MS medium containing 5 g/liter of the carbon sources (or 2 g/liter sodium propionate) plus 5 g/liter DTDP as precursor substrate. After 4 days of incubation, the PMP content of each sample was analyzed. The error bars represent the standard deviations from three independent experiments.

Fig 5

Relationship between cell amount and PMP production. (A) Growth curves of different strains. Symbols: ●, growth curve of strain SHX5 in the first cultivation stage; ○, growth curve of strain SHX13 in first cultivation stage; ■, OD₆₀₀ of strain SHX5; □, OD₆₀₀ of strain SHX13. Arrowheads indicate the time points when the cells were transferred from the first to the second stage. (B) PMP content relative to cell growth in the first stage. Black bars, strain SHX5; white bars, strain SHX13. The error bars represent the standard deviations from three independent experiments.

Fig 6

PMP content, OD, cell dry weight, and supernatant analysis during the time course of the cultivation experiment. A. mimigardefordensis strains SHX5 and SHX13 were first incubated in modified MS medium containing 2 g/liter NH₄Cl and 7 g/liter glycerol at the first cultivation stage. After their OD had reached 500 Klett units, all cells were transferred into the same MS medium containing 7 g/liter glycerol plus 9 g/liter DTDP as a precursor. (A) Increase of OD (Klett unit) and cell dry weight in the second cultivation stage. Symbols: ● and ○, turbidity increase of SHX5 and SHX13; ▲ and △, cell dry weight increase of SHX5 and SHX13. (B) PMP content over time in the second-stage cultivation stage. Symbols: ★ and ☆, strains SHX5 and SHX13. (C) Concentrations of DTDP, 3MP, and glycerol in different culture supernatants in the second-stage cultivation stage. Symbols: ▼ and ▽, consumption of DTDP in cultures of SHX5 and SHX13; ■ and □, degradation of glycerol in cultures of SHX5 and SHX13; ♦ and ♢, occurrence of 3MP in cultures of strains SHX5 and SHX13.

Fig 7

PMP production in engineered strains. Cells were first incubated in modified MS medium containing 2 g/liter NH₄Cl, 10 g/liter glycerol, and 0.4 g/liter yeast extract when their OD₆₀₀ had reached about 3 to 4 (late exponential phase). Then the cells were collected and transferred into fresh medium supplied with 6 g/liter DTDP as precursor. After 4 days of incubation, all samples were analyzed by GC to determine the PMP content. The error bars represent the standard deviations from three independent experiments.

Fig 8

PMP production in cells of A. mimigardefordensis strain SHX22 cultivated in the presence of different glycerol concentrations. The strains were first incubated in modified MS medium with 2 g/liter NH₄Cl, 10 g/liter glycerol, and 0.4 g/liter yeast extract. After the cells had reached the early stationary growth phase (OD₆₀₀ of approximately 4.2 to 4.4), they were collected and transferred to fresh medium supplied with different concentrations of glycerol (10, 20, 30, 40, and 60 g/liter) and 6 g/liter DTDP as a precursor. After 4 days of incubation, all samples were analyzed for PMP content by GC analysis.