Simultaneous Improvements of Pseudomonas Cell Growth and Polyhydroxyalkanoate Production from a Lignin Derivative for Lignin-Consolidated Bioprocessing

Abstract

Cell growth and polyhydroxyalkanoate (PHA) biosynthesis are two key traits in PHA production from lignin or its derivatives. However, the links between them remain poorly understood. Here, the transcription levels of key genes involved in PHA biosynthesis were tracked in Pseudomonas putida strain A514 grown on vanillic acid as the sole carbon source under different levels of nutrient availability. First, enoyl-coenzyme A (CoA) hydratase (encoded by phaJ4) is stress induced and likely to contribute to PHA synthesis under nitrogen starvation conditions. Second, much higher expression levels of 3-hydroxyacyl-acyl carrier protein (ACP) thioesterase (encoded by phaG) and long-chain fatty acid-CoA ligase (encoded by alkK) under both high and low nitrogen (N) led to the hypothesis that they likely not only have a role in PHA biosynthesis but are also essential to cell growth. Third, 40 mg/liter PHA was synthesized by strain A_phaJ4C1 (overexpression of phaJ4 and phaC1 in strain A514) under low-N conditions, in contrast to 23 mg/liter PHA synthesized under high-N conditions. Under high-N conditions, strain A_alkKphaGC1 (overexpression of phaG, alkK, and phaC1 in A514) produced 90 mg/liter PHA with a cell dry weight of 667 mg/liter, experimentally validating our hypothesis. Finally, further enhancement in cell growth (714 mg/liter) and PHA titer (246 mg/liter) was achieved in strain A_{xyl_alkKphaGC1} via transcription level optimization, which was regulated by an inducible strong promoter with its regulator, XylR-P_xylA, from the xylose catabolic gene cluster of the A514 genome. This study reveals genetic features of genes involved in PHA synthesis from a lignin derivative and provides a novel strategy for rational engineering of these two traits, laying the foundation for lignin-consolidated bioprocessing.IMPORTANCE With the recent advances in processing carbohydrates in lignocellulosics for bioproducts, almost all biological conversion platforms result in the formation of a significant amount of lignin by-products, representing the second most abundant feedstock on earth. However, this resource is greatly underutilized due to its heterogeneity and recalcitrant chemical structure. Thus, exploiting lignin valorization routes would achieve the complete utilization of lignocellulosic biomass and improve cost-effectiveness. The culture conditions that encourage cell growth and polyhydroxyalkanoate (PHA) accumulation are different. Such an inconsistency represents a major hurdle in lignin-to-PHA bioconversion. In this study, we traced and compared transcription levels of key genes involved in PHA biosynthesis pathways in Pseudomonas putida A514 under different nitrogen concentrations to unveil the unusual features of PHA synthesis. Furthermore, an inducible strong promoter was identified. Thus, the molecular features and new genetic tools reveal a strategy to coenhance PHA production and cell growth from a lignin derivative.

Keywords: Pseudomonas putida; lignin-consolidated bioprocessing; polyhydroxyalkanoate synthesis.

FIG 1

Proposed metabolic pathways for lignin-to-PHA bioconversion. Key genes and metabolic intermediates are highlighted in red. Identifications (IDs) of the corresponding genes in A514 are shown. (A) Lignin degradation pathways. Dashed arrows indicate multiple steps. (B) De novo fatty acid biosynthesis. (C) β-Oxidation pathway. After each turn of the cycle, an acyl-CoA (indicated as C_n) that is two carbons shorter than the initial one (indicated as C_{n + 2}) is generated. The dashed line connects acyl-CoA intermediates of different chain lengths. (D) PHA biosynthesis pathways. (E, F, and G) Expression ratios for alkK homologs and phaG and phaJ genes, as well as for PHA synthetic genes, in A514 that were grown under the low-N conditions, compared to those grown under the high-N conditions. The ratios that were >1 were defined as induced. (H) Expression levels of the phaJ4, phaG, and alkK (PputA514_1839) genes in A514 under both high-N and low-N conditions. The inset in panel H shows the expression level of phaJ4 at an appropriate scale. The expression levels and expression ratios are defined in Materials and Methods.

FIG 2

Effect of the regulator, regulator-induced expression time, and inducer concentrations on the activity of the promoter P_xylA. (A) Organization of the putative A514 xylose catabolism locus, as well as reporter constructs used to detect activity of P_xylA. P, promoter; gfp, green fluorescent protein gene. (B) Activities of P_xylA and P_van under various inducing conditions. For bars 1 to 5, either 0 mM or 0.2 mM (final concentration) xylose was added when strains were at the mid-exponential phase. After 6 h of induction, green fluorescence intensity was measured to determine the promoter activity. Bars 6 and 7 represent the P_van activity in A_Pvan at the mid-exponential phase and the stationary phase, respectively. Bars 8 and 9 represent the XylR-P_xylA activity in A_R-PxylA at the stationary phase, which was exposed to 0 mM or 2 mM xylose for 12 h, respectively. pTT, pPROBE-TT; Con, concentration. (C) Time point-dependent induction of XylR-P_xylA activity. Response of XylR-P_xylA to different growth phases at which 0.2 mM xylose (final concentration) was added. Fluorescence intensity was measured every 3 h until A_R-PxylA entered the stationary phase. (D) Xylose concentration-dependent induction of XylR-P_xylA activity. A_R-PxylA at the mid-exponential phase was exposed to different concentrations of xylose for 6 h. For panels B, C, and D, all of the strains were cultivated in M9 medium supplemented with 15 mM vanillic acid. Fluorescence intensity, which was detected to determine the promoter activity, is expressed in arbitrary units normalized for 10⁶ CFU.