The yeast Kluyveromyces lactis has been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressible LAC4 promoter (P _LAC4 ) is the most widely used promoter to drive recombinant protein expression in K. lactis However, P _LAC4 is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, P _LAC4 is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novel K. lactis promoter system that is both strong and tightly controllable. We first tested several different endogenous K. lactis promoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P₃₅₀) was created by combining segments of two K. lactis promoters, namely, the strong constitutive P _GAP1 promoter and the carbon source-sensitive P _ICL1 promoter. We demonstrate that P₃₅₀ is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P₃₅₀-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P₃₅₀ hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins in K. lactisIMPORTANCE The yeast Kluyveromyces lactis is an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P₃₅₀) for use in K. lactis P₃₅₀ is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be "autoinduced" at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

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