Fermentative Production of Cysteine by Pantoea ananatis

Abstract

Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe.IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production.

Keywords: Pantoea ananatis; amino acid fermentation; cysteine; cysteine desulfhydrase; cysteine efflux.

FIG 1

Biosynthetic pathway and regulation of cysteine synthesis in Pantoea ananatis and related bacteria. The major metabolites and enzymes responsible for cysteine biosynthesis from glucose and thiosulfate or sulfate are indicated by solid lines. The key regulatory effects provided by feedback inhibition of SAT (encoded by cysE) and 3-PGDH (encoded by serA) by cysteine and l-serine, respectively, as well as transcriptional regulation by the master regulator of sulfur metabolism, CysB, are indicated by dotted lines. NAS, N-acetylserine; OAS, O-acetylserine; PAPS, 3-phosphoadenosine 5-phosphosulfate; 3-PG, 3-phosphoglycerate; 3-PGDH, 3-phosphoglycerate dehydrogenase; SAT, serine acetyltransferase; Sbp, sulfate-binding protein; Ser, serine.

FIG 2

Resistance to cysteine. Escherichia coli MG1655 strains carrying each plasmid containing a series of genes identified as sources of cysteine resistance were challenged by 200 μM cysteine in M9 medium. Representative growth curves are shown for the strains carrying the vector (pSTV-29, ○), leuE (pSTV-leuE, ■), eamA (pSTV-eamA, ◻), ccdA (pSTV-ccdA, ●), cefA (pSTV-PA36ccd, △), and cefB (pSTV-cefB, ▲) without cysteine (A) and with 200 μM cysteine (B).

FIG 3

Effects of CysM overexpression on CcdA activation and cysteine production. The production of cysteine in the medium (gray bars) and activity of CysM (O-acetylserine sulfhydrylase B) based on the crude cell extract (white circles) from each CysM-enhanced variant (AG6181, AG6180, and AG6184) constructed by engineering cysteine-producing Pantoea ananatis strain AG4854 are shown (top). The images of native PAGE and activity staining of the ccdA product obtained from the crude cell extract of each of the CysM variants are shown (bottom). Values represent the averages based on the results from four independent experiments, and the error bars represent one standard deviation.

FIG 4

Effects of enhancing the efflux pumps on the fermentative production of cysteine in an engineered producer strain AG6184. Each gene encoding the cysteine efflux pumps was introduced into AG6184 using pACYC177 (vector) driven by the original promoters (A) or pMIV-5JS (vector) driven by P_nlp0 (B). Productive cultivation was performed under standard conditions and using standard medium, except that glucose was supplemented at 60 g · liter⁻¹ instead of the usual 40 g · liter⁻¹, and cultivation was terminated at 28 h when approximately 40 g · liter⁻¹ glucose had been consumed (within approximately 10% of the error range) (A), or under standard conditions and using standard medium with regular 40 g · liter⁻¹ glucose, and termination of cultivation at 22 h when 40 g · liter⁻¹ glucose had been consumed (within approximately 10% of the error range) (B). A transcriptional regulator, cefR, was cointroduced with cefA by use of pACYC-PA36ccd to completely enhance the effects of cefA. Values represent the averages based on the results from four independent experiments, and the error bars represent one standard deviation.